Super-resolution imaging reveals resistance to mass transfer in functionalized stationary phases*

Chemical separations are costly in terms of energy, time, and money. Separation methods are optimized with inefficient trial-and-error approaches that lack insight into the molecular dynamics that lead to the success or failure of a separation and, hence, ways to improve the process. We perform super-resolution imaging of fluorescent analytes in four different commercial liquid chromatography materials. Surprisingly, we observe that chemical functionalization can block over 50% of the material's porous interior, rendering it inaccessible to small molecule analytes. Only in situ imaging unveils the inaccessibility when compared to the industry-accepted ex situ characterization methods. Selectively removing some of the functionalization with solvent restores pore access without significantly altering the single-molecule kinetics that underlie the separation and agree with bulk chromatography measurements. Our molecular results determine that commercial “fully porous” stationary phases are over-functionalized and provide a new avenue to characterize and direct separation material design from the bottom-up.